Initiation of RNA synthesis from DNA templates by RNA polymerase (RNAP) is a multi-step process, in which initial recognition of promoter DNA by RNAP triggers a series of conformational changes in ...both RNAP and promoter DNA. The bacterial RNAP functions as a molecular isomerization machine, using binding free energy to remodel the initial recognition complex, placing downstream duplex DNA in the active site cleft and then separating the nontemplate and template strands in the region surrounding the start site of RNA synthesis. In this initial unstable “open” complex the template strand appears correctly positioned in the active site. Subsequently, the nontemplate strand is repositioned and a clamp is assembled on duplex DNA downstream of the open region to form the highly stable open complex, RP
o. The transcription initiation factor, σ
70, plays critical roles in promoter recognition and RP
o formation as well as in early steps of RNA synthesis.
RbpA and CarD are essential transcription regulators in mycobacteria. Mechanistic analyses of promoter open complex (RPo) formation establish that RbpA and CarD cooperatively stimulate formation of ...an intermediate (RP2) leading to RPo; formation of RP2 is likely a bottleneck step at the majority of mycobacterial promoters. Once RPo forms, CarD also disfavors its isomerization back to RP2. We determined a 2.76 Å-resolution crystal structure of a mycobacterial transcription initiation complex (TIC) with RbpA as well as a CarD/RbpA/TIC model. Both CarD and RbpA bind near the upstream edge of the -10 element where they likely facilitate DNA bending and impede transcription bubble collapse. In vivo studies demonstrate the essential role of RbpA, show the effects of RbpA truncations on transcription and cell physiology, and indicate additional functions for RbpA not evident in vitro. This work provides a framework to understand the control of mycobacterial transcription by RbpA and CarD.
Transcription initiation requires formation of the open promoter complex (RPo). To generate RPo, RNA polymerase (RNAP) unwinds the DNA duplex to form the transcription bubble and loads the DNA into ...the RNAP active site. RPo formation is a multi-step process with transient intermediates of unknown structure. We use single-particle cryoelectron microscopy to visualize seven intermediates containing Escherichia coli RNAP with the transcription factor TraR en route to forming RPo. The structures span the RPo formation pathway from initial recognition of the duplex promoter in a closed complex to the final RPo. The structures and supporting biochemical data define RNAP and promoter DNA conformational changes that delineate steps on the pathway, including previously undetected transient promoter-RNAP interactions that contribute to populating the intermediates but do not occur in RPo. Our work provides a structural basis for understanding RPo formation and its regulation, a major checkpoint in gene expression throughout evolution.
Display omitted
•Cryo-EM structures of 7 intermediates in promoter opening pathway from RPc to RPo•Intermediates populated by using an inhibitor and a promoter with unstable RPo•RNAP and DNA conformational changes in mobile regions mark the steps in the pathway•Transient interactions identified in intermediates are not found in RPc or RPo
Cryo-EM structures of RNA polymerase-promoter DNA intermediates identify stages in transcription initiation from the initial recognition of double-stranded promoter DNA in RPc to final promoter melting in RPo. Structural analyses of RNA polymerase and DNA conformational changes delineate steps in the pathway. Biochemical and genetic characterization support their functional importance.
Noncoding small RNAs regulate gene expression in all organisms, in some cases through direct association with RNA polymerase (RNAP). Here we report that the mechanism of 6S RNA inhibition of ...transcription is through specific, stable interactions with the active site of Escherichia coli RNAP that exclude promoter DNA binding. In fact, the DNA-dependent RNAP uses bound 6S RNA as a template for RNA synthesis, producing 14-to 20-nucleotide RNA products (pRNA). These results demonstrate that 6S RNA is functionally engaged in the active site of RNAP. Synthesis of pRNA destabilizes 6S RNA-RNAP complexes leading to release of the pRNA-6S RNA hybrid. In vivo, 6S RNA-directed RNA synthesis occurs during outgrowth from the stationary phase and likely is responsible for liberating RNAP from 6S RNA in response to nutrient availability.
We present an approach for preparing cryo-electron microscopy (cryo-EM) grids to study short-lived molecular states. Using piezoelectric dispensing, two independent streams of ~50-pl droplets of ...sample are deposited within 10 ms of each other onto the surface of a nanowire EM grid, and the mixing reaction stops when the grid is vitrified in liquid ethane ~100 ms later. We demonstrate this approach for four biological systems where short-lived states are of high interest.
The first step in gene expression in all organisms requires opening the DNA duplex to expose one strand for templated RNA synthesis. In
, promoter DNA sequence fundamentally determines how fast the ...RNA polymerase (RNAP) forms "open" complexes (RPo), whether RPo persists for seconds or hours, and how quickly RNAP transitions from initiation to elongation. These rates control promoter strength in vivo, but their structural origins remain largely unknown. Here, we use cryoelectron microscopy to determine the structures of RPo formed de novo at three promoters with widely differing lifetimes at 37 °C: λP
(t
∼10 h), T7A1 (t
∼4 min), and a point mutant in λP
(λP
) (t
∼2 h). Two distinct RPo conformers are populated at λP
, likely representing productive and unproductive forms of RPo observed in solution studies. We find that changes in the sequence and length of DNA in the transcription bubble just upstream of the start site (+1) globally alter the network of DNA-RNAP interactions, base stacking, and strand order in the single-stranded DNA of the transcription bubble; these differences propagate beyond the bubble to upstream and downstream DNA. After expanding the transcription bubble by one base (T7A1), the nontemplate strand "scrunches" inside the active site cleft; the template strand bulges outside the cleft at the upstream edge of the bubble. The structures illustrate how limited sequence changes trigger global alterations in the transcription bubble that modulate the RPo lifetime and affect the subsequent steps of the transcription cycle.
Recent advances in single-particle cryogenic electron microscopy (cryo-EM) have enabled the structural determination of numerous protein assemblies at high resolution, yielding unprecedented insights ...into their function. However, despite its extraordinary capabilities, cryo-EM remains time-consuming and resource-intensive. It is therefore beneficial to have a means for rapidly assessing and optimizing the quality of samples prior to lengthy cryo-EM analyses. To do this, we have developed a native mass spectrometry (nMS) platform that provides rapid feedback on sample quality and highly streamlined biochemical screening. Because nMS enables accurate mass analysis of protein complexes, it is well suited to routine evaluation of the composition, integrity, and homogeneity of samples prior to their plunge-freezing on EM grids. We demonstrate the utility of our nMS-based platform for facilitating cryo-EM studies using structural characterizations of exemplar bacterial transcription complexes as well as the replication-transcription assembly from the SARS-CoV-2 virus that is responsible for the COVID-19 pandemic.
Display omitted
•A native MS-based diagnostic and screening platform for cryo-EM samples•Provides rapid assessment of sample stability and homogeneity•Enables iterative biochemical screening for optimizing sample conditions for cryo-EM•Applied to structural studies of transcription complexes from bacteria and SARS-CoV-2
A major bottleneck in single-particle cryo-EM involves sample preparation and assessment of sample stability and homogeneity. Olinares et al. have developed a time-saving native mass spectrometry-based platform that provides rapid feedback on sample quality and enables highly streamlined biochemical screening for optimal sample conditions prior to cryo-EM analysis.
Recent biophysical studies of mycobacterial transcription have shed new light on this fundamental process in a group of bacteria that includes deadly pathogens such as Mycobacterium tuberculosis ...(Mtb), Mycobacterium abscessus (Mab), Mycobacterium leprae (Mlp), as well as the nonpathogenic Mycobacterium smegmatis (Msm). Most of the research has focused on Mtb, the causative agent of tuberculosis (TB), which remains one of the top ten causes of death globally. The enzyme RNA polymerase (RNAP) is responsible for all bacterial transcription and is a target for one of the crucial antibiotics used for TB treatment, rifampicin (Rif). Here, we summarize recent biophysical studies of mycobacterial RNAP that have advanced our understanding of the basic process of transcription, have revealed novel paradigms for regulation, and thus have provided critical information required for developing new antibiotics against this deadly disease.
Noncovalent self-assembly of biopolymers is driven by molecular interactions between functional groups on complementary biopolymer surfaces, replacing interactions with water. Since individually ...these interactions are comparable in strength to interactions with water, they have been difficult to quantify. Solutes (osmolytes, denaturants) exert often large effects on these self-assembly interactions, determined in sign and magnitude by how well the solute competes with water to interact with the relevant biopolymer surfaces. Here, an osmometric method and a water-accessible surface area (ASA) analysis are developed to quantify and interpret the interactions of the remarkable osmolyte glycine betaine (GB) with molecular surfaces in water. We find that GB, lacking hydrogen bond donors, is unable to compete with water to interact with anionic and amide oxygens; this explains its effectiveness as an osmolyte in the Escherichia coli cytoplasm. GB competes effectively with water to interact with amide and cationic nitrogens (hydrogen bonding) and especially with aromatic hydrocarbon (cation−π). The large stabilizing effect of GB on lac repressor−lac operator binding is predicted quantitatively from ASA information and shown to result largely from dehydration of anionic DNA phosphate oxygens in the protein−DNA interface. The incorporation of these results into theoretical and computational analyses will likely improve the ability to accurately model intra- and interprotein interactions. Additionally, these results pave the way for development of solutes as kinetic/mechanistic and thermodynamic probes of conformational changes and formation/disruption of molecular interfaces that occur in the steps of biomolecular self-assembly processes.
Though opening of the start site (+1) region of promoter DNA is required for transcription by RNA polymerase (RNAP), surprisingly little is known about how and when this occurs in the mechanism. ...Early events at the λPR promoter load this region of duplex DNA into the active site cleft of Escherichia coli RNAP, forming the closed, permanganate-unreactive intermediate I₁. Conversion to the subsequent intermediate I₂ overcomes a large enthalpic barrier. Is I₂ open? Here we create a burst of I₂ by rapidly destabilizing open complexes (RPo) with 1.1 M NaCl. Fast footprinting reveals that thymines at positions from -11 to +2 in I₂ are permanganate-reactive, demonstrating that RNAP opens the entire initiation bubble in the cleft in a single step. Rates of decay of all observed thymine reactivities are the same as the I₂ to I₁ conversion rate determined by filter binding. In I₂, permanganate reactivity of the +1 thymine on the template (t) strand is the same as the RPo control, whereas nontemplate (nt) thymines are significantly less reactive than in RPo. We propose that: (i) the +1(t) thymine is in the active site in I₂; (ii) conversion of I₂ to RPo repositions the nt strand in the cleft; and (iii) movements of the nt strand are coupled to the assembly and DNA binding of the downstream clamp and jaw that occurs after DNA opening and stabilizes RPo. We hypothesize that unstable open intermediates at the λPR promoter resemble the unstable, transcriptionally competent open complexes formed at ribosomal promoters.
PDF
